Title:Adventures of the Purple Fluorescent Protein pGEM-gbr22
Introduction: To produce a recombinant protein, a researcher is faced with a variety of choices as to where to start. To facilitate decision-making, we describe a consensus ‘what to try first’ strategy based on collective analysis of the expression and purification of over 10,000 different proteins [1]. This is where the basis on the foundation of any research begins. [1] The bacteria Escherichia coli has been a common expression host for the production of various soluble proteins [1]. These proteins are used all throughout biological and biomedical sciences [1]. This lab emphasizes three significant aspects of a protein, expression, purification and characterization. The strain E. coli, BL21(DE3) (New England BioLabs, Ipswich, MA), is used as the expression host and transformed by the plasmid pGEM gbr-22, which encodes for a fluorescent protein (Not fluorescent) that exhibits a purple color found from the the coral in theGreat Barrier Reef. If proper protein production techniques are done correctly, then you could estimate that the protein is purple and fluorescent and come to the conclusion of the weight of your protein, which is approximately 26 kDa. Weak hypothesis
Materials & Methods: Part 1: Expression It would be good if you added a little intro sentence here talking about the 3 steps. For expression, E. Coli BL21(DE3) was transferred into 2 transformation tubes. pGEM-gbr22 plasmid was added to the DNA tube. Both tubes were heat shocked at 42 degrees Celsius for 45 seconds. SOC media was added to each tube and were shaken in the incubator for 30 minutes at 37 degrees Celsius. 50ul of bacteria/SOC mixture was pipetted from each tube and added to plates containing ampicillin. Explain about selecting for ampicillin resistance Overnight incubation followed. A single colony of bacteria was then added to LB/ampicillin media. The two tubes were incubated for 8 hours at 37 degrees Celsius. The starter culture containing pGEM-gbr22 was transferred into an Erlenmeyer flask that contained LB/ampicillin. The flask was put into a shaking incubator for 24 hours. A sample of culture was taken (sample 1). The remaining content of the bacteria were poured into a conical tube and centrifuged into an Allegra X-15 (Beckman Coulter, Inc., Brea, CA) and set to run for 10 minutes (4 degrees Celsius). The purple pellet at the bottom was saved Why?and remaining liquid was disposed. 2.5 mL of 1x PBS solution was mixed with the pellet. Lysozyme was added to the tube where it’s stored in the -20 degrees Celsius freezer.
Part 2: Purification
For purification, 2uL of Cyanase was added to the tube and stored in the -20 degrees Celsius freezer. <------ this is extra info. you don't need itThe lysate was distributed into two tubes and centrifuged. Then, supernatant was obtained (sample 2). The supernatant from both tubes were transferred into a conical tube and filtered through a syringe filter. An Econo column was used to run the supernatant along with Ni-NTA resin/buffer. The supernatant was mixed with Ni-NTA resin/buffer and run through the column (Sample 3). 5mL of Wash elution that consisted of 1x PBS and 20mM imidazole was run through the column (Sample 4). Then, an elution buffer that consisted of 1x PBS and 250mM imidazole was run through twice (first time: sample 5; second time sample 6). The absorbance of the protein solution from Elution 1 was measured through Nanodrop spectrophotometry at 280 nanometers in order to estimate the yield of the protein. 6-HIS Tag? how is the protein separated?
Part 3: Characterization
For characterization, gel electrophoresis is used. From previous labs, six samples were gathered and placed into a heat block at 95 degrees Celsius for 5 minutes, after which they were centrifuged for 2 minutes. Then, loaded into Bio-Rad precast gel. The SDS electrophoresis was prepared, so the samples could run for twenty-five minutes in a Mini-PROTEAN tank filled with 500mL of 5x TGS buffer. The samples were suspended in 6x sample loading buffer and loaded into the gel along with MW standard (Page Ruler; Prestained Protein Ladder, ThermoScientific). After running for 25 minutes, the gel was stained using Imperial protein stain and rinsed with NP water in a shaker overnight. The next day, the gel was dried on Whatman paper and covered with wrap at 75 degrees Celsius for 1.5 hours. (talk about the samples)
Results:
Figure 1: Positive control plate of DNA with BL21, the bacterial cell with pGEM-gbr22, which is the plasmid of the transformation exhibited after a 24 hour incubation period at 37 degrees Celsius.
Figure 2: A random bacterial growth of saliva, cheek cells, and mouth bacteria on ampicillin before the 24 hour incubation period.
Figure 3: Negative control plate with BL21 (DE3) bacterial cell and no plasmid.
Figure 4: Erlenmeyer flask of a large culture of BL21 (DE3) bacterial cell that has been transformed with plasmid DNA pGEM-gbr22 after a single colony has been cultivated in LB broth & ampicillin.
Figure5: A wet pellet of 0.21 grams obtained from centrifuging a large culture of BL21 (DE3) bacterial cells transformed with plasmid DNA, pGEM-gbr22 from Figure 1.
Figure6: 5 mL of Elution 1 buffer obtained with purified protein gbr22.
Figure 7: 5 mL of Elution 2 buffer obtained with purified protein gbr22.
Figure8: Absorbance vs. wavelength reading for Elution 1 at a wavelength of 280 nm(nanometers) for protein gbr22 on trial 1 (n=2).
Beer's Law Calculation:Concentration determined of protein using 280 nm wavelength
A=Ebc
(.211)=(38,850)(1)c
c= 5.43 X 10^-6 mol/L
(5.43 X 10^-6 mol/L)(25794.2 g/mol)= 0.140 g/L or 0.140 mg/mL
YieldDetermination:At 280 nm wavelength
(0.140 mg/mL)(5 mL)= .7 mg
Figure 9: Molecular Weight Standard (Thermo Scientific PageRuler Prestained Protein Ladder; 4-20% Tris-glycine gel (SDS)-PAGE; Product #26616)
Figure 10: Destained gel after 24 hours on orbital shaker with molecular weight standard in lane 2 & Samples 1-6 are loaded in lanes 3-8.
Figure 11: Dried gel after an hour and a half in gel dryer where Molecular Weight Standard is in lane 2 & Samples 1-6 are loaded in lanes 3-8.
Discussion:
The results above exhibit the demonstration of protein expression, purification, and characterization techniques applied determining the success of our purple fluorescent protein analysis. Lysozyme was added at the end of the protein expression lab to the solution containing E. coli cells in order to digest the cell wall. Then, the benzonase/cyanase was added during the purification lab because it was needed to digest the DNA/RNA of the E. coli. Both of these were added to help separate the protein from all of the insignificant matter that isn't necessary for process of obtaining our protein. For the importance of the purification process, the HIS6x tag system works by the protein that has been modified to have six histidine residues added to the C-terminus, which can be used to separate the protein from other proteins. These residues will bind to the nickel that is immobilized on a column matrix such as Ni-NTA resin. Then, the protein is released from the Ni-NTA resin by adding imidazole, which competes with the histidine residues for this binding. The affinity of the 6xHis-tagged protein for nickel ions is the basis of purification.
The Samples were obtained throughout each of the three labs, for they all contribute to the end result for our protein characterization. Sample 1 was obtained in expression lab where the E. coli cells are present with proteins and other cell debris. Sample 2 was obtained in the purification lab where it only contained a soluble fraction after the lysozyme was added. Sample 3 is the Ni-NTA resin flow through the column after the syringe filtering where this sample was collected after letting the protein go through and contained proteins that did not stick to the nickel causing the bands to be similar. Sample 4 was collected after the Wash solution was run through and contained proteins that were loosely bound to the Ni-NTA resin where only a few proteins remain. Sample 5 was collected after the Elution 1 buffer was run through where it contained the purple protein and had one dark band indicating the purple protein, which is where only 1 band should be present in lane 7 of Figure 10. Sample 6 was collected after the Elution 1 buffer, which becomes Elution 2 as it was run through a second time where extra imidazole is added so it is where the leftover protein remains.
As seen in figure 10, several other lighter bands were found in lane 7, which contains Elution 1 indicating that the purity of the sample was not 100%. This leaves estimated purity of the final protein to be 30%. When comparing the band to the molecular weight standard, the band corresponded to approximately 26 kDa.
Many of the possible sources of error include the creation of our imidazole in the first solution lab, where it could have been made incorrectly causing for the contamination of Elution 1. Also, the difference in each obtained sample where the other protein bands present in lane 7 of Figure 10 where the purity of the protein is estimated can be because it is possible that other proteins have Histidine rich regions that will bind to the nickel. This leads to where the purification step could have been done incorrectly. Good Analysis!
Conclusions:
In the first part of this lab, bacterial cells BL21(DE3) were transformed with the plasmid (pGEM-br22), which shows the overexpression of the purple protein. After, follows the lab where the protein was extracted from the bacterial cell as it was lysed in order to release the soluble protein through purification. Then, the protein is purified by the affinity tag & Ni-NTA resin where centrifugation release the insoluble cell debris after, as samples were obtained through this process. This significant, tedious process leads to where the samples were then run through gel electrophoresis through characterization.This essential part in characterization of the protein leads to the future implications of enzyme assay analysis where the activity of the protein will be measured.
References: [1] Graslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; Protein production and purification. Nature Methods.2008, 5(2): 135-46.
[2] http://www.embl.de/pepcore/pepcore_services/index.html
Introduction:
To produce a recombinant protein, a researcher is faced with a variety of choices as to where to start. To facilitate decision-making, we describe a consensus ‘what to try first’ strategy based on collective analysis of the expression and purification of over 10,000 different proteins [1]. This is where the basis on the foundation of any research begins. [1] The bacteria Escherichia coli has been a common expression host for the production of various soluble proteins [1]. These proteins are used all throughout biological and biomedical sciences [1]. This lab emphasizes three significant aspects of a protein, expression, purification and characterization. The strain E. coli, BL21(DE3) (New England BioLabs, Ipswich, MA), is used as the expression host and transformed by the plasmid pGEM gbr-22, which encodes for a fluorescent protein (Not fluorescent) that exhibits a purple color found from the the coral in the Great Barrier Reef. If proper protein production techniques are done correctly, then you could estimate that the protein is purple and fluorescent and come to the conclusion of the weight of your protein, which is approximately 26 kDa. Weak hypothesis
Materials & Methods:
Part 1: Expression
It would be good if you added a little intro sentence here talking about the 3 steps.
For expression, E. Coli BL21(DE3) was transferred into 2 transformation tubes. pGEM-gbr22 plasmid was added to the DNA tube. Both tubes were heat shocked at 42 degrees Celsius for 45 seconds. SOC media was added to each tube and were shaken in the incubator for 30 minutes at 37 degrees Celsius. 50ul of bacteria/SOC mixture was pipetted from each tube and added to plates containing ampicillin. Explain about selecting for ampicillin resistance Overnight incubation followed. A single colony of bacteria was then added to LB/ampicillin media. The two tubes were incubated for 8 hours at 37 degrees Celsius. The starter culture containing pGEM-gbr22 was transferred into an Erlenmeyer flask that contained LB/ampicillin. The flask was put into a shaking incubator for 24 hours. A sample of culture was taken (sample 1). The remaining content of the bacteria were poured into a conical tube and centrifuged into an Allegra X-15 (Beckman Coulter, Inc., Brea, CA) and set to run for 10 minutes (4 degrees Celsius). The purple pellet at the bottom was saved Why?and remaining liquid was disposed. 2.5 mL of 1x PBS solution was mixed with the pellet. Lysozyme was added to the tube where it’s stored in the -20 degrees Celsius freezer.
Part 2: Purification
For purification, 2uL of Cyanase was added to the tube and stored in the -20 degrees Celsius freezer. <------ this is extra info. you don't need itThe lysate was distributed into two tubes and centrifuged. Then, supernatant was obtained (sample 2). The supernatant from both tubes were transferred into a conical tube and filtered through a syringe filter. An Econo column was used to run the supernatant along with Ni-NTA resin/buffer. The supernatant was mixed with Ni-NTA resin/buffer and run through the column (Sample 3). 5mL of Wash elution that consisted of 1x PBS and 20mM imidazole was run through the column (Sample 4). Then, an elution buffer that consisted of 1x PBS and 250mM imidazole was run through twice (first time: sample 5; second time sample 6). The absorbance of the protein solution from Elution 1 was measured through Nanodrop spectrophotometry at 280 nanometers in order to estimate the yield of the protein. 6-HIS Tag? how is the protein separated?
Part 3: Characterization
For characterization, gel electrophoresis is used. From previous labs, six samples were gathered and placed into a heat block at 95 degrees Celsius for 5 minutes, after which they were centrifuged for 2 minutes. Then, loaded into Bio-Rad precast gel. The SDS electrophoresis was prepared, so the samples could run for twenty-five minutes in a Mini-PROTEAN tank filled with 500mL of 5x TGS buffer. The samples were suspended in 6x sample loading buffer and loaded into the gel along with MW standard (Page Ruler; Prestained Protein Ladder, ThermoScientific). After running for 25 minutes, the gel was stained using Imperial protein stain and rinsed with NP water in a shaker overnight. The next day, the gel was dried on Whatman paper and covered with wrap at 75 degrees Celsius for 1.5 hours. (talk about the samples)
Results:
Beer's Law Calculation: Concentration determined of protein using 280 nm wavelength
A=Ebc
(.211)=(38,850)(1)c
c= 5.43 X 10^-6 mol/L
(5.43 X 10^-6 mol/L)(25794.2 g/mol)= 0.140 g/L or 0.140 mg/mL
Yield Determination: At 280 nm wavelength
(0.140 mg/mL)(5 mL)= .7 mg
Discussion:
The results above exhibit the demonstration of protein expression, purification, and characterization techniques applied determining the success of our purple fluorescent protein analysis.
Lysozyme was added at the end of the protein expression lab to the solution containing E. coli cells in order to digest the cell wall. Then, the benzonase/cyanase was added during the purification lab because it was needed to digest the DNA/RNA of the E. coli. Both of these were added to help separate the protein from all of the insignificant matter that isn't necessary for process of obtaining our protein. For the importance of the purification process, the HIS6x tag system works by the protein that has been modified to have six histidine residues added to the C-terminus, which can be used to separate the protein from other proteins. These residues will bind to the nickel that is immobilized on a column matrix such as Ni-NTA resin. Then, the protein is released from the Ni-NTA resin by adding imidazole, which competes with the histidine residues for this binding. The affinity of the 6xHis-tagged protein for nickel ions is the basis of purification.
The Samples were obtained throughout each of the three labs, for they all contribute to the end result for our protein characterization. Sample 1 was obtained in expression lab where the E. coli cells are present with proteins and other cell debris. Sample 2 was obtained in the purification lab where it only contained a soluble fraction after the lysozyme was added. Sample 3 is the Ni-NTA resin flow through the column after the syringe filtering where this sample was collected after letting the protein go through and contained proteins that did not stick to the nickel causing the bands to be similar. Sample 4 was collected after the Wash solution was run through and contained proteins that were loosely bound to the Ni-NTA resin where only a few proteins remain. Sample 5 was collected after the Elution 1 buffer was run through where it contained the purple protein and had one dark band indicating the purple protein, which is where only 1 band should be present in lane 7 of Figure 10. Sample 6 was collected after the Elution 1 buffer, which becomes Elution 2 as it was run through a second time where extra imidazole is added so it is where the leftover protein remains.
As seen in figure 10, several other lighter bands were found in lane 7, which contains Elution 1 indicating that the purity of the sample was not 100%. This leaves estimated purity of the final protein to be 30%. When comparing the band to the molecular weight standard, the band corresponded to approximately 26 kDa.
Many of the possible sources of error include the creation of our imidazole in the first solution lab, where it could have been made incorrectly causing for the contamination of Elution 1. Also, the difference in each obtained sample where the other protein bands present in lane 7 of Figure 10 where the purity of the protein is estimated can be because it is possible that other proteins have Histidine rich regions that will bind to the nickel. This leads to where the purification step could have been done incorrectly.
Good Analysis!
Conclusions:
In the first part of this lab, bacterial cells BL21(DE3) were transformed with the plasmid (pGEM-br22), which shows the overexpression of the purple protein. After, follows the lab where the protein was extracted from the bacterial cell as it was lysed in order to release the soluble protein through purification. Then, the protein is purified by the affinity tag & Ni-NTA resin where centrifugation release the insoluble cell debris after, as samples were obtained through this process. This significant, tedious process leads to where the samples were then run through gel electrophoresis through characterization.This essential part in characterization of the protein leads to the future implications of enzyme assay analysis where the activity of the protein will be measured.
References:
[1] Graslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; Protein production and purification. Nature Methods.2008, 5(2): 135-46.
[2] http://www.embl.de/pepcore/pepcore_services/index.html